Clostridium difficile is a Gram-positive anaerobe that is the cause of enteric disease in many animal species including humans. In humans, C. difficile associated diarrhea (CDAD) is a commonly diagnosed cause of hospital-associated and antimicrobial-associated diarrhea. With the emergence of the hypervirulent NAP1/027 strains in hospitals globally, a sharp increase in mortality rates has been observed (Kaier and Frank, Antimicrob Agents Chemother 2009, 53 (10), 4574-4575). While previous reports of C. difficile epidemics were restricted to single institutions or wards, more recently reports of a wider distribution of outbreaks are increasing (Bignardi et al, J Hosp Infect 2008, 70 (1), 96-98). Infection with C. difficile can lead to severe diarrhea, abdominal pain and further complications such as pseudomembranous colitis, inflammation and ulceration of the lining of the intestinal wall.
Current practice for the treatment of CDAD is the administration of antibiotics. Metronidazole, vancomycin, and fidaxomicin are among the most commonly-used antibiotics for treatment of CDAD. However, these approaches can only be used once the patient has contracted CDAD, and may be inefficient in the face of a drug-resistant bacterium. Additionally, the relapse rate of successfully treated patients is approximately 20%.
In light of the emergence and increasing severity of CDAD, there has been a significant increase in the number of research articles on C. difficile detection and characterization of virulence factors and toxins. However, to date little attention has been paid to the surface carbohydrate-containing molecules produced by this emerging pathogen. An early study by Poxton and Cartmill, J. Gen Micro 1982, 128, 1365-1370, described the characterization of two cell surface antigens extracted from the bacterial cell surface. Twenty years ago, the identification of a capsular polysaccharide (CPS)-like structure by electron microscopy was reported (Baldassarri et al, Microbiologica 1991, 14 (4), 295-300) followed by a detailed characterization of a C. difficile CPS (Ganeshapillai et al, Carbohydr. Res. 2008, 343 (4), 703-710). A recent publication demonstrated that the flagellin protein from a number of C. difficile clinical isolates was glycosylated with a novel O-linked glycan (Twine et al, 2009, 191(22), 7050-7062). In general however, the surface polysaccharides of the genus Clostridium are relatively poorly understood. Although there is information relating to C. perfringens CPS structures (Kalelkar et al, 1997, 299 (3), 119-128), the Clostridium genus is diverse genetically and it is unlikely that surface polysaccharides are conserved across the genus.
With respect to CPS of C. difficile, the work of Ganeshapillai, supra showed that a ribotype 027 strain produced two polysaccharides; the first polysaccharide (PSI) was a branched pentaglycosyl phosphate repeat unit composed of [→4-α-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3]-α-D-Glcp-(1→2)-α-D-Glcp-(1→P→] while the second polysaccharide (PSII) consisted of a hexaglycosyl phosphate repeat unit with the structure [→6)-β-D-Glcp-(1→3)-β-D-GalpNAc-(1→4)-α-D-Glcp-(1→4)-[β-D-Glcp(1→3]-β-D-GalpNAc-(1→3)-α-D-Manp-(1→P→]. The authors also confirmed the presence of the latter structure on the surface of two other C. difficile isolates; however, they also acknowledged that further investigations regarding the use of the structures in immune response were warranted.
Others (Oberli et al, Chem Biol, 2011, 18 (5), 580-588; Danieli et al, Org Letters. 2011, 13 (3), 378-381; Monteiro et al, WO 2009/033268) have-investigated vaccines that target the PSII CPS. Oberli, supra, and Danieli, supra, both use a synthetic monomeric structure to target the CPS; however, this may not provide a good mimic of the natural epitopes present on the polymers on the pathogen. Monteiro shows limited cross-reactivity of the PSII polysaccharide. To date, no further vaccines have been reported against C. difficile surface polysaccharides.
Another strategy is a therapeutic approach against the C. difficile toxin. This involves the use of antibodies (e.g., monoclonal antibodies) or antibody fragments (e.g., single-domain antibodies) specific for the toxin (for example, Hussack et al, 2011 JBC 286 (11), pp. 8961-8976). However, this anti-toxin strategy does not kill the bacteria, but rather only neutralizes the toxin, leaving the bacteria intact.
Thus, it is clear to those of skill in the art that there remains a profound need to establish a conserved and broadly cross-reactive, immunogenic antigen in order to develop a safe and effective vaccines for conferring immunity to patients at risk for developing C. difficile infections.